Project Summary: The long-term goal of this project is to develop an understanding of the cellular and molecular mechanisms of human cytomegalovirus (HCMV) latency in CD34+ hematopoietic progenitor cells (HPCs) and the effect of latent and reactivating virus on hematopoiesis. The ability of HCMV to reactivate in CD34+ HPCs is exquisitely linked to HPC differentiation and hematopoiesis. Understanding how HCMV regulates hematopoiesis is crucial to identifying mechanisms of latency and reactivation as well as disease. HCMV is a species-specific virus that establishes a persistent/latent infection in the host after primary infection. Our group and others have shown that HCMV encodes several microRNAs (miRNAs), small 21-24 base pair single-stranded RNA species that regulate gene expression and multiple cellular processes involved in the virus life-cycle. In preliminary work we have observed that two HCMV latently expressed miRNAs up-regulate and one expressed during lytic replication down-regulates Early Growth Response Gene-1 (Egr-1) that is a transcription factor induced by MEK/ERK signaling that regulates the maintenance human progenitor cells (HPC) or ?Stemness? in the bone marrow through the induction and suppression of key cytokines and co-stimulatory molecules. Deletion of these two HCMV miRNAs that up-regulate Egr-1 results in a virus that fails to reactivate in vitro or in a humanized mouse model. Additionally, we have shown that the miRNAs that regulate Egr-1 signaling pathway alter HPC myelopoiesis suggesting link between HPC latency and reactivation and HPC myelopoiesis. We also observe that Egr-1 up-regulates UL138 expression a gene shown to maintain HCMV latency. Lastly, we show that HCMV latent infection of HPC up-regulates expression of TGF-??that is regulated by Egr-1. Latent HCMV infection of HPC inhibits myelopoiesis in vitro that is restored by neutralization of TGF-?. We hypothesize that HCMV miRNAs that up-regulate Egr-1 maintain CD34+ HPC stemness and latency through expression of UL138 and miRNAs that down-regulate Egr-1 induce reactivation thus providing a regulatory switch for viral latency or reactivation. In the first aim we will identify and characterize additional HCMV miRNA signaling targets that regulate Egr-1 and their role in viral latency and reactivation. In the second aim we will characterize HCMV miRNA regulation of Egr-1 mediated UL136 and UL138 expression in latency. In the last aim we will characterize HCMV miRNA regulation of Egr-1 and TGF-? on HPC stemness and myelopoiesis using both in vitro systems and a unique humanized mouse model. These studies have significant implications on HCMV induced myelosupression observed in bone marrow transplant patients.